1. Field of the Invention
This invention relates to a method for a synthesis of anhydrothrombin. More particularly, this invention relates to a method for a highly efficient and convenient synthesis of anhydrothrombin having a specific ability as a ligand to be utilized for the technique of affinity chromatography which is effectively adopted for separating and refining antithrombin III, blood coagulation factors VIII and XIII, etc.
2. Description of Related Art
The antithrombin III, one species of the glycoprotein which belongs to an .alpha..sub.2 -globulin in blood plasma, discharges an important role of inhibiting the reaction of blood coagulation or adjusting a reaction of coagulation in the blood vessel by reacting with thrombin or an activating factor and forming a corresponding complex.
As respects the method for separating and purifying the antithrombin of this quality, the technique of affinity chromatography which effects the separation by virtue of the specific affinity for a heparin (ligand) is at an advantage in (1) simplifying the operation of purification, (2) allowing satisfactory separation of extraneous substances, and (3) enjoying a satisfactory activity recovery ratio.
When this technique is used for isolating the antithrombin III from a side fraction obtained by the cold ethanol technique which is widely practiced on a commercial scale, however, the product occurs in very low yields. When this substance is isolated from heat-treated blood plasma, the product likewise arises in low yields. This poor efficiency of the isolation of the antithrombin III may be logically explained by a supposition that since the technique of affinity chromatography using heparin relies on the structure in the proximity of the lysine residue, a site for the bondage of antithrombin III to heparin, the structure of this portion is vulnerable to the low-temperature ethanol treatment or the heat treatment. It is, therefore, some other structural site in the antithrombin III that deserves utility for the technique of affinity chromatography. The use of the structure in the proximity of the residue as the center of arginine reaction, therefore, is recommendable. Further, the heparin is not free from virus. The antithrombin III which has been isolated, therefore, has the possibility of being infected with a virus. The circumstances, therefore, have urged the need to search for some other virus-free ligand.
As a measure of the solution of such faults as mentioned above with a view to the points enumerated above, JP-B-59-7,694 and the report of Tomono et al. published in ACTA Haematologica Japonica, Vol. 49, No. 4, 969, 1986 have proposed the use of an inactivated thrombin which, as a virus-free ligand alternative to heparin, reacts with the antithrombin III and exhibits affinity for a covalent bond complex without inducing formation of the complex and, therefore, provides a method for enabling the affinity chromatography utilizing the structure in the proximity of the residue as the center of the arginine reaction of antithrombin III to effect highly efficient fractionation of the antithrombin III contained in the blood plasma and the blood plasma-protein mixture such as of the cold ethanol fraction. Further, the patent specification and the text of the report mentioned above describe examples of the synthesis of anhydro-thrombin as an inactivated thrombin from thrombin by the process of synthesis (conventional process A) schematically depicted in FIG. 1. A review of these examples reveals that the reaction for anhydridization requires the reaction system to be adjusted with an alkali to pH 9.0.
The mechanism of the reaction of anhydridization which has been heretofore attained popularly by inactivating the serine residue of such other protein as trypsin or chymotrypsin with a varying sulfonyl fluoride such as phenylmethane sulfonyl fluoride (PMSF) and then treating the inactivated serine residue with an alkali thereby depriving this residue of the PMS (phenylmethane sulfonyl group) modifying the protein is reported in "J. Biochem., 81, 647-656, 1977," "J. Biochem., 81, 657-663, 1977," "Biochemical and Biophysical Research Communications, Vol. 47, No. 6, 1972," and "Biochemical and Biophysical Research Communications, Vol. 46, No. 4, 1972," for example. These reports have statements that in unison purport to demonstrate that the reaction of the anhydridization of the active serine residue in such protein as trypsin or chymotrypsin is allowed to proceed by retaining the modifying protein such as PMSF in a high range of pH (not lower than pH 11). As concerns the anhydridization of thrombin, the aforementioned statement in literature that the reaction proceeds even when the treatment with an alkali is performed in a range of pH (pH 9.0) lower than the range of pH proper for trypsin or chymotrypsin may well deserve attention. Incidentally, the conventional process A taught in the literature mentioned above avoids performing the anhydridization of thrombin in the high range of pH (not lower than pH 11). This avoidance of the high range of pH is logically explained by a supposition that the thrombin cannot be utilized as a ligand because it is not stable in such a high range of pH as fits the trypsin or chymotrypsin and, when subjected at all to the alkali treatment at a pH of not lower than 11, undergoes coagulation and insolubilization and, if permitted to undergo an anhydrodization, will not be enabled to refold it.
Dr. Ashton of the U.S., in his recent report in "Biochemistry 1995, 34, 6454-6463, offers a statement that his replication of the process of synthesis (the conventional process A) which avoids anhydrodizing thrombin in a high range of pH (not lower than pH 11) as disclosed in the literature mentioned above has failed to attain synthesis of the anhydrothrombin, while granting that no simple comparison is allowed because he has used the thrombin originating in bovine blood serum in the place of refined human thrombin (.alpha.-thrombin originating in Cohn Paste III).
Apart from this assertion, Dr. Ashton describes in the same literature his success in synthesizing the anhydrothrombin owing to the use of guanidine hydrochloride (Gdn-HCl) during the course of reaction indicated in the process of synthesis (conventional process B) schematically illustrated in FIG. 1 for the purpose of precluding the thrombin from coagulation and insolubilization in the high range of pH.
The conventional process B, however, is deficient in practicability because the procedure thereof is complicated, the duration of synthesis thereof is elongated, and the yields in which the anhydrothrombin is produced thereby are extremely low (21% as shown in the data of the literature) as compared with the other processes.
An object of this invention, therefore, is to find a solution of the faults mentioned above and consequently provide a method for the synthesis of an anhydrothrombin which shortens the duration of synthesis, facilitates the procedure, and heightens the yields in which the anhydrothrombin is produced.
The conventional process B shown in FIG. 1 accomplishes the synthesis of an anhydrothrombin by using Gdn-HCl for depriving the thrombin (protein) of hydrophobicity and solubilizing the modified PMS-thrombin. It is suspected that the Gdn-HCl is used for solubilizing the nonpolar residue when the stereostructure of the protein is collapsed by the denaturation due to a change in pH and the nonpolar side chain is consequently exposed to the surface. The addition of the Gdn-HCl which functions as a denaturing agent naturally causes further denaturation of the thrombin and nevertheless brings about successful synthesis of the anhydrothrombin finally by virtue of refolding. The thrombin is unstable as compared with the trypsin and, under the alkaline conditions necessary for the anhydridization, assumes a denatured state which is expressed as .DELTA.G (denatured free energy)&lt;0. It is believed that the conventional process B uses the Gdn-HCl for the purpose of precluding the occurrence of association and coagulation in this state.